The present invention provides substrates on which a microarray of molecules, such as a microarray of oligonucleotides, can be formed, and further provides methods for generating such microarrays.
Microarrays of hundreds or thousands of biological compounds are widely utilized for biological analysis. Tiny droplets, each containing a different known reagent, usually distinct polynucleotide or polypeptide biopolymers, such as, known DNA fragments, can be deposited and immobilized in a regular array on a solid substrate, such as a glass microscope slide. The array of reagents can be exposed to an analyte solution containing unknown target sequences, for example, complementary DNA (cDNA) fragments, pre-labeled with fluorescent or radioactive chemical labels. Under certain conditions, binding reactions or hybridizations occur between a polynucleotide sequence in the array and a complementary sequence in the analyte. Subsequent optical or radiosensitive scanning procedures can be used to determine those position(s) in the microarray at which hybridization reactions have occurred, thereby identifying the complementary compounds present in the solution.
Various methods for immobilizing synthesized oligonucleotides or peptides on a substrate surface via chemical reactions are known. Such methods typically attach linking groups to a substrate surface at pre-defined locations, and connect known nucleotide or amino acid sequences to these linking groups, in order to create microarrays. One drawback of such methods is that the oligonucleotides or peptides in the microarrays are immobilized in particular configurations, which may inhibit optimal interactions between target sequences in test samples and the immobilized oligonucleotides or peptides.
Another known method of generating microarrays utilizes agarose gel pads placed at predefined locations on a flat surface. In one application of such microarrays, oligonucleotides of a known length and sequence are placed within each gel pad along with a test sample containing the target sequence. Each gel pad provides a three-dimensional environment in which the oligonucleotides and the target sequence in the test sample can react.
In many of the known methods for forming microarrays, the interactions between the immobilized molecules and their complementary counterparts can be hindered by the presence of linking groups, and/or the specific orientations of the immobilized molecules. Further, even in microarrays that utilize three-dimensional gel pads, although the oligonucleotides are not immobilized, the interactions between the oligonucleotides within the gel and the test sample are not optimal because they occur within the confines of a gel pad rather than in an aqueous solution.
In addition, conventional microarrays have limited densities largely as a result of the shortcomings of the methods utilized for generating them. For example, fixation of reagents on a substrate to form a microarray can require the presence of large bulky protective groups that often limit the density of the array. Likewise, when agarose gel pads are employed for forming a microarray, there is an upper limit for the density of gel pads on a substrate.
Accordingly, a need exists for microarrays in which optimal biological reactions can occur between reagents deposited in, or on, a micro-location of the array and a target molecule in a test sample. A need also exists for microarrays having higher densities of micro-locations suitable for containing reagents for biological reactions.